Cells infected with plus-strand RNA viruses undergo dramatic remodeling of their intracellular membranes into so-called replication organelles. The functional properties of the replication membranes that are required to support viral RNA replication for plus-strand viruses are unknown. Our recent work demonstrated that RNA viruses manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication which are distinct in protein and lipid composition from that of the host. We found that enteroviral 3A protein, hijacks host phosphatidylinositol-4-kinase III? (PI4KIII?) to membranes to generate organelles enriched in phosphatidylinositol-4-phosphate (PI4P) lipids. We discovered that the PI4P-rich lipid membrane microenvironment is essential for both enteroviral and flaviviral RNA synthesis, and PI4KIII? enzymes are critical for generating this lipid microenvironment. Furthermore we found that enteroviral RNA polymerases specifically and preferentially bind PI4P lipids. Our findings have brought forward a new paradigm to virology; revealing how viruses can selectively exploit specific elements of the host to form specialized organelles where host phosphoinositide lipids are key to the regulation of viral RNA replication. Based on these findings we hypothesize that PI4P lipids are critical regulators of enteroviral RNA polymerase activity and link membrane reorganization with optimized viral RNA replication in vivo. Our specific aims are: to determine the mechanisms by which PI4P lipids regulate enteroviral RNA synthesis and to identify the PI4P lipid-binding domain on enteroviral RNA polymerases. Using biochemical, spectroscopic, genetic and computational methods we will determine the location of PI4P binding site on the RNA polymerase and whether PI4P binding can modulate the enzymatic activity of the polymerase and other viral proteins within the replication complex. This will further our understanding of all positive strand RNA viral infection which depend on membranes for RNA synthesis; to elucidate the mechanisms by which enteroviral 3A protein selectively promotes recruitment of host PI4KIII?. Using a variety of biochemical, genetic and imaging methods we will test whether PI4KIII? is hijacked directly by 3A or through intermediate host factors. This information will be important for designing and targeting drugs to block this critical event; to determine the nanoscale association of viral and host components with PI4P lipid enriched replication organelles using quantitative super-resolution light-based imaging. Using Photoactivated Light Microscopy (PALM), a single molecule super-resolution imaging method, we will investigate the membrane structure of PI4P-lipid enriched replication organelles and determine the spatial organization and density of replication complexes. This will provide high-resolution spatiotemporal information on the coupling between membrane dynamics and viral RNA replication. Collectively these studies will address an important new paradigm in virology, providing insight into how phosphoinositide lipids can regulate viral RNA replication. These findings will have far reaching implications for designing new therapeutics to combat viral infections.